Effect of combined praziquantel and recombinant glutathione S-transferase on resistance to reinfection in murine Schistosomiasis mansoni.

This study was undertaken to evaluate the effect of recombinant Schistosoma mansoni-26 Glutathione S-transferase (rSm 26 GST) or soluble egg antigen (SEA) alone and in addition to praziquantel (PZQ) on the state of resistance to S. mansoni reinfection. The associated changes in the immune responses were evaluated. The experimental group of mice were injected intravenously before S. mansoni infection (80 cercariae/mouse) either with rSm26 GST (1 microgx4) or SEA (10 microgx4) in addition to PZQ (2x500 mg/kg) administered 6 weeks post-infection. Seven control groups were used, three of them were the infected (80 cercariae/mouse), the challenged (240 cercariae/mouse) and the infected challenged controls (80+240 cercariae/mouse). The rest of the four groups were the treated controls receiving: the GST-Lyzate, rSmGST, SEA and PZQ in the same doses and at the same timings. Challenge infection was conducted for all the groups 8 weeks post-infection. Animals were sacrificed 3 weeks post-challenge. After sacrifice animals were perfused and percentage resistance to reinfection was calculated. Immune responses were assessed by the measurement of hepatic granuloma diameter, intralesional T-cell phenotypes and serum immunoglobulin isotypes. The highest percentage of resistance to reinfection was observed in rGST-treated group while the lowest percentage of resistance was detected in PZQ-treated group. Whereas in mice receiving combined rGST or SEA and PZQ, percentage resistance to reinfection was significantly higher than that in PZQ treated mice. The remarkable reduction in granuloma diameter in rGST-treated group with or without PZQ was associated with decrease in the intralesional L(3)T(4)(+) and increase in Lyt(2)(+) T-cell phenotypes. However, no special relationship was observed between the percentage of resistance and the changes in granuloma diameter or intralesional T-cell phenotypes. The increase in percentage resistance to reinfection was found accompanied by increased anti SWAP IgE. Combined rGST and PZQ provided the complementary goals of improved state of resistance to reinfection 'which was compromized after cure with PZQ' and the maximal reduction in granuloma diameter.

Immunolocalization of macrophage adhesion molecule-1 and macrophage inflammatory protein-1 in schistosomal soluble egg antigen-induced granulomatous hyporesponsiveness.

This work studied the histopathological changes and the changes in the expression of macrophage adhesion molecule-1 (Mac-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) in a murine model of soluble egg antigen (SEA) - induced granulomatous hyporesponsiveness. Histopathological results of hepatic sections in an SEA group showed early acceleration of ova destruction and markedly diminished granuloma cellularity with eosinophils and macrophages still being the predominant cells. Later, giant cells and pigmented macrophages that were scattered among granuloma cells and in intimate contact with the deposited eggs were more predominant in the SEA group than in the infected control group. Concurrently, the counts of Mac-1 positive cells were significantly increased in liver sections of the SEA group than the infected control group during the course of infection. MIP-1alpha showed early higher counts followed by lower counts in the later stages of infection on granuloma cells in the SEA group than the infected control group. During the course of infection, similar distribution of Mac-1 and MIP-1alpha was present in both groups. This study suggests that sensitization with SEA probably leads to enhancement of phagocytic activity of macrophages via increasing expression of Mac-1 and hence engulfment of ic3b coated schistosomal products such as ova. It leads to rapid destruction of ova and hence decreases the host inflammatory response to infection and amelioration of hepatic pathology which would be a promising approach in reduction of host morbidity and mortality.

Parasite-specific antibody profile in human fascioliasis: application for immunodiagnosis of infection.

The antibody isotype response to an adult Fasciola worm antigen preparation (FWAP) was examined in sera from 60 Egyptians with parasitologically confirmed fascioliasis by an ELISA. The FWAP-specific IgG1 and IgG4 antibodies were found in 97-100% of the patients. The ratio of the mean absorbance values between infected patients and healthy controls was 9.7 and 29.7 for IgG1 and IgG4 antibodies, respectively. The IgM, IgA, IgG2, and IgG3 antibodies were less dominant. In contrast to IgG1 antibodies, which were often detected in sera from patients infected with Schistosoma, Echinococcus granulosus, Ascaris lumbricoides, Ancylostoma duodenale, or Hymenolepis nana, FWAP-specific IgG4 antibodies were detected exclusively in the sera of patients with fascioliasis. The data thus support the conclusion that an IgG4/ELISA with crude FWAP as antigen may be used for sensitive and accurate immunodiagnosis of human fascioliasis.

Effect of early treatment with praziquantel on serum connective tissue metabolite markers in children and adolescents with intestinal schistosomiasis mansoni.

This work was designed to assess the reflection of early treatment by praziquantel (CAS 55268-74-1, EMBAY 8440, Biltricide) on serum connective tissue metabolite markers (hyaluronic acid and procollagen III peptide) in patients with active intestinal schistosomiasis. Children and adolescent subjects from primary and secondary schools in an endemic area of schistosomiasis mansoni were included. Age-matched subjects from an urban area served as normal controls. All subjects were examined clinically and parasitologically. Detection of hepatitis B seromarkers was also done. The infected subjects were treated with praziquantel at a dose of 60 mg/kg of body weight which was repeated after 4 weeks. Serum hyaluronic acid and procollagen III peptide were measured by radioimmunoassay. High hyaluronic acid was encountered in infected subjects when compared to their respective age-matched controls. Significant decrease of 4 and 8 weeks post-treatment was noted when compared to ist level before treatment. There was no significant change in serum procollagen III peptide on comparing infected subjects to their controls, whereas a significant increase was observed in its level after 4 and 8 weeks post-treatment compared to that before treatment. This work suggests that early treatment of intestinal schistosomiasis with specific chemotherapy (praziquantel) decreases serum hyaluronic acid and increases procollagen III peptide probably via downregulation of granulomatous inflammatory cell reaction and activation of collagenase enzymes, respectively.

Efficacy of an antipathology vaccine in murine schistosomiasis administered with and without chemotherapy.

This study was undertaken to study the efficacy of praziquantel (PZQ) in potentially tolerized Schistosoma mansoni infected, egg-injected C57BL/6 mice, receiving multiple administrations of soluble egg antigen (SEA) intravenously (i.v.). Four animal groups were studied. Experimental group I received four injections of SEA (10 micrograms) intravenously on days -7, -5, -3 and -2 before infection and PZQ orally (500 mg/kg over two consecutive days 7 weeks post-infection. Three control groups received the following treatment: group II received the same tolerizing dose of SEA without PZQ, group III received PZQ in the same dose and at the same timing. Group IV received S. mansoni infection and egg injection 8 weeks post-infection and served as an infected, egg-injected control. Egg injection was conducted 8 weeks post-infection using viable S. mansoni eggs via the tail vein. Animals were killed 16 days post-egg injection, i.e. 10 weeks post-infection. After sacrifice, lungs and livers were removed for histopathological study and measurement of granuloma diameters. Spleens and serum were collected for the assay of lymphoproliferative response to SEA and antischistosomal immunoglobulins. The worm and egg burdens were also studied. Compared to infected, egg-injected untreated controls, repeated i.v. administrations of SEA down-regulated egg-injected (pulmonary) and egg-deposited (hepatic) granulomas and the lymphoproliferative response to SEA. Antischistosomal IgG level was increased. Susceptibility to S. mansoni infection was not found to be different from that in the infected, egg-injected controls. PZQ in the dose used caused complete eradication of worms, disappearance of immature egg stages, decrease in the number of mature eggs and an increase in the number of dead eggs. Hepatic granuloma diameter, lymphoproliferative response to SEA and IgG level were reduced. In mice receiving a combined regimen of multiple SEA administrations and PZQ with down-regulated granuloma and reduced lymphoproliferative response to SEA, the efficacy of PZQ was the same as in mice receiving PZQ alone. This was shown by comparable grades of worm and egg reduction. The histopathological examination of liver and lung sections in the different treated groups revealed moderate to small-sized hypocellular granulomas. Although no statistically significant difference was recorded between the mean granuloma diameters of the experimental group receiving both the tolerizing dose of SEA and PZQ compared to the group receiving the tolerizing dose of SEA without chemotherapy, this experimental group showed the least associated histopathological parenchymal changes. It appears from this work that combined SEA and PZQ provided many complementary goals; a reduction of egg-induced pathology, minimal parenchymal changes and the eradication of worms.

Immunoregulatory potential of exogenous Schistosoma mansoni soluble egg antigen in a model of experimental schistosomiasis--I. Regulation of granuloma formation in vivo.

This study was undertaken to assess the optimum conditions required to reduce the vigorous host granulomatous reaction around Schistosoma mansoni eggs. Soluble schistosomal egg antigen (SEA) at a concentration of 10 or 100 micrograms protein was administered i.p. or i.v. into unprimed C57BL/6 mice. SEA was injected either alone or in combination with cyclophosphamide (CY) 100 or 50 mg/kg via i.p. route. Seven or 14 days later viable eggs of S. mansoni were injected via the tail vein into treated groups and untreated normal controls. Mice were sacrificed 8, 16 and 24 days after the injection of eggs. The lungs were removed for histopathological study, measurement of granuloma diameter and phenotypic analysis of granuloma intralesional T-cell subsets. Compared to untreated controls, the lower concentration of SEA (10 micrograms) administered by the i.v. route 7 days before egg injection, induced a significant reduction in granuloma diameter 16 days after egg injection than that by the i.p. route or at a higher SEA concentration (100 micrograms). Compared to untreated controls, the higher dose of CY (100 mg/kg), given i.p. alone or in combination with 10 micrograms SEA by the i.v. or i.p. route, induced a significant reduction in granuloma diameter, while 50 mg/kg CY did not cause any reduction. The reduction in granuloma diameter by i.v. administration of low SEA concentration alone or in combination with CY IP, was associated with a decrease in the granuloma intralesional L3T4+/Lyt2+ ratio. The decrease in the ratio was due to an increase in Lyt2+ cells. The results suggest that the use of low dose SEA by the i.v. route alone or combined with an immunosuppressive drug ameliorates pathological changes concurrent with S. mansoni infection.

Evaluation of specific Fasciola antigen in the immunodiagnosis of human fascioliasis in Egypt.

Enzyme-linked immunosorbent assay (ELISA) and enzyme linked immunoelectrotransfer blot technique (EITB) were employed for the detection of circulating Fasciola antibodies in infected human sera using a specific Fasciola antigen, prepared by immunoaffinity purification of homogenates of Fasciola hepatica adult worms. Ninety two individuals diagnosed clinically and parasitologically were classified into: Fascioliasis group (21 patients), schistosomiasis group (21 patients) and subjects harbouring other parasitic infections (50 patients). Eighteen healthy individuals served as normal controls. ELISA was 100% sensitive and 93% specific with 96.5% diagnostic efficacy, whereas EITB was 100% sensitive and specific with 100% diagnostic efficacy. Our data revealed that ELISA can be used as a good screening test while EITB can serve as a confirmatory test for immunodiagnosis of fascioliasis.

Purification and characterization of a specific Fasciola antigen.

Specific Fasciola antigen was prepared from homogenates of Fasciola hepatica adult worms. The homogenate was ultracentrifuged and the supernatant containing crude Fasciola antigen was then passed over a cyanogen bromide activated sepharose 4B column coupled with antiserum against Schistosoma mansoni adult worm surface antigen. The specific, Schistosoma-free Fasciola antigen was tested for its specificity by immunodiffusion. Characterization of the specific Fasciola antigen was done by gradient poly-acrylamide gel electrophoresis and immunoblotting technique. The electrophoresis migration pattern of specific Fasciola antigen, stained with Coomassie blue, showed 7 bands in the 12-54 kDa regions. Using the immunoblotting technique, a batch of positive fascioliasis sera recognized two specific bands at the 33 and 54 kDa regions.

Evaluation of different immunodiagnostic techniques for diagnosis of hydatidosis in Egypt.

In this study detection of the circulating hydatid antibodies was performed by three serological tests: Immunoelectrophoresis (IEP), Enzyme linked immunosorbent assay (ELISA) and Enzyme linked immunoelectrotransfer blot (EITB) technique. The antigen which was used in these tests was Echinococcus granulosus antigen free of host (camel) antigen by immunoaffinity chromatography. Most serum samples were pre-selected from the patients on the basis of clinical syndrome or past history of possible diagnosis of echinococcosis. In addition, physical aids to diagnosis, especially sonographic and plain X-ray examination have already been carried out. The hydatidosis group was subgrouped according to the site of the cysts in different organs. The surgical removal of hydatid cysts was taken as a sure diagnosis of hydatid disease. The outcome of comparison between the three serological tests in surgically confirmed cases, showed that ELISA technique is a good screening test (88.2% sensitivity, 88.8% specificity and 88.5% diagnostic efficacy) and EITB is a good confirmatory test (100% sensitivity, 100% specificity and 100% diagnostic efficacy).

Identification and characterization of specific hydatid antigen fraction(s).

A specific hydatid antigen was prepared in this study from Echinococcus granulosus cyst in livers and lungs of camels. Elimination of host "camel" protein from crude hydatid fluid was achieved by two methods: Salting out using ammonium sulfate precipitation method and immunoaffinity purification using coupled anticamel antibody to cyanogenbromide activated sepharose 4B gel. Testing the prepared hydatid antigen against anticamel serum, using immunodiffusion method, indicated that the affinity purified hydatid antigen was almost completely purified from camel protein. Characterization of the affinity purified hydatid antigen, using immunoelectrophoresis, showed positive arc 5 precipitation when tested against known positive antihydatid sera. Further characterization with gradient gel electrophoresis, showed with silver stain that the dominant and most consistently demonstrable proteins occurred as a complex in the 52/62 KDa region. Strong reaction with the 52/62 KDa complex was consistently observed when the affinity purified hydatid antigen was probed with known positive reference antihydatid sera. The identified hydatid antigen fraction(s) with 52/62 KDa complex can provide promising non-invasive parameter for diagnosis of Hydatidosis.

Detection of circulating anodic antigen before and after specific chemotherapy in experimental murine Schistosomiasis mansoni.

In two groups of mice infected with 60 (group I) and 120 (group II) Schistosoma mansoni cercariae, respectively, the effects of intensity and duration of infection, and of praziquantel therapy (curative vs subcurative dose) on the levels of circulating anodic antigen (CAA), were studied. CAA was measured in trichloracetic acid-treated serum samples with an avidin-biotin enzyme-linked immunosorbent assay (AB-ELISA) using the monoclonal anti-CAA antibody. Total worm burdens, oogram patterns and ova counts/g liver and intestine were followed up. The lowest detectable level of CAA was about 1.0 ng/ml, and was positive with a worm load of 3-5/mouse. CAA levels became already detectable as early as 1-2 weeks post-infection (pi) before any parasitological parameter and showed a significant drop from the 11th-12th week pi onwards. A positive correlation was demonstrated between the CAA level and worm load. Following successful praziquantel therapy, CAA disappeared earlier than any of the other parameters studied.

Aminoterminal propeptide of type III procollagen: a marker of disease activity in schistosomal patients.

The serum concentration of aminoterminal propeptide of type III procollagen was measured in 44 Egyptian healthy controls and 29 patients with hepatosplenomegaly originating from endemic areas for schistosomiasis in Egypt. Patients were classified into two main groups according to the histopathological pattern of the liver biopsy: patients with active schistosomal liver fibrosis and patients with inactive schistosomal liver fibrosis. Serum aminoterminal propeptide of type III procollagen levels were elevated in most of patients with active fibrosis but not in those with inactive schistosomiasis. From the present work, it is suggested that aminoterminal propeptide of type III procollagen can be used as a marker for active fibrogenesis in patients with schistosomal liver fibrosis.

Effect of praziquantel on hepatic fibrosis in experimental Schistosomiasis mansoni.

The effect of specific chemotherapy on hepatic fibrosis was studied in Swiss albino mice infected with Schistosoma mansoni. The effect of treatment with praziquantel at 8, 12, and 20 weeks postinfection on fibrosis was assessed by determination of total hepatic collagen content, ratio of type III/I + III collagen, granuloma volume, and histopathological examination of liver section. Total collagen content was reduced in mice treated at the 8th week of infection compared to respective infected controls. The ratio of type III/I collagen was within normal limits. The decrease in collagen deposition was not observed when treatment was given 12 or 20 weeks postinfection. Early specific treatment of schistosomiasis may be important in the therapeutic approach to the control of morbidity in schistosomiasis.

Effect of praziquantel on certain immune responses of schistosomal Egyptian patients. I. Changes of specific immunoglobulins.

Specific antischistosomal IgG, IgM, and IgE were estimated by ELISA in 117 rural school students before specific treatment with praziquantel monthly for 3-4 months thereafter. IgG and IgM were estimated as percentage of bound antibodies. IgE was estimated by avidin-biotin ELISA (AB-ELISA) as IU/ml using a panel of known IgE standards. Soluble surface Schistosoma mansoni adult worm antigen was used for all estimates. Total IgE was estimated in a smaller group by an ELISA kit. The percentage of specific IgE was calculated. A group of endemic controls (22 students) and non-endemic controls (17 cases) were included. Statistical analysis of results showed the specific immunoglobulins to be significantly reduced 2 months after treatment of the schistosomal cases. These reduced levels, however, were still significantly higher than those of controls. The presence of early hepatosplenomegaly and the co-existence of other parasites had no significant influence on the results. No correlation could be established between the levels of specific antischistosomal IgG, M and E and the intensity of infection. The significance of these results is discussed.